9. The CRISPR CAS System
• Cas9genes
• non-coding RNA elements called CRISPR RNA(crRNA)
• Small trans-encoded CRISPR RNA i.e., trans-activating crRNA
(tracrRNA).
The two RNA sequences crRNA and tracrRNA forms a complex
known as guide RNA, which determines the specificity of the
cleavage of the target sequence in the nucleic acid along with the
Protosapcer Adjacent Motif (PAM), a 5’-NGG sequence.
• The Cas9 protein is an endonuclease associated with CRISPR loci, which is responsible for the double-stranded
breaks (DSBs) at the site, when targeted by a guide RNA.
• Cleavage occurs on both strands (scissors) 3 bp upstream of the PAM.
www.mirusbio.com
10. • 6 distinct types of bacterial CRISPR systems identified so far.
• Type II is the basis for current genome engineering applications
Addison V. Wright, et al. 2016, Cell
12. Sternberg S H, et al. March, 2014, Nature, 507(7490):62-67.
PAM recognition regulates Cas9 nuclease activity
13. • Programmable nucleases composed of two components, which must be expressed in
cells to perform genome editing; the Cas9 nuclease and an engineered single guide
RNA (sgRNA).
• sgRNA has 20 nucleotides at the 5’ end that directs Cas9 to the complementary
target site.
• Any DNA sequence of the form N20-NGG can be targeted by altering the first 20
nucleotides of the gRNA for novel genome editing applications.
• The Cas9 endonuclease consists of two different domains, which includes a large
globular recognition (REC) specific functional domain, connected to a smaller
nuclease (NUC) domain. The NUC domain further accommodates two nuclease
sites, RuvC and HNH, and also a PAM-interacting site.
• Cas9 undergoes a conformational change upon gRNA binding that shifts the
molecule from an inactive, non-DNA binding conformation, into an active DNA-
binding conformation.
RNA-guided engineered nucleases (RGENs)
14. Khatodia et al. 2016. Frontiers in Plant Science
The Cas9 nuclease induces double stranded breaks (DSB) at the target site which can be repaired either by Non-
Homologous End Joining method or Homologous Recombination by cellular system which results in gene disruption
by indels or gene addition/correction, respectively.
15. • Cas9 Nickase (a mutation in native Cas9)
-Cas9 nickase with a RuvC or HNH mutation has the ability to create a nick, instead of a DSB at the target site. The
individual nicks in the genome can be typically repaired with high fidelity homology-directed repair (HDR).
- has improved the specificity and helped mitigate the off-target phenomena
• Inactive dCas9
-The nuclease deficient catalytically inactive mutant version of Cas9 (dCas9) has been used for RNA-guided transcription
regulation, instead of genome editing.
-This modified system has been used for CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) for highly
efficient and precise gene silencing and activation.
-The dCas9 has also been used to eliver GFP to targeted genomic locations (Anton et al., 2014).
• Dimeric RNA-Guided FokI Nucleases (RFNs)
-Dimeric RNA-guided FokI Nucleases (RFNs) is a fusion of a catalytically inactive dCas9 protein with the FokI nuclease
domain.
-Dimerization of two RFNs rather than co-localization is required for efficient genome editing activity, which gives a plus
point over the Cas9 nickase for high genome editing frequencies and reduced off-target mutations.
Native Cas9 Protein Variants
16. Truncated Guide RNAs (truRNA)
sgRNA variants with shorter regions of target complementarities (17 nucleotides) .
Ribozyme-gRNA-Ribozyme (RGR)
An artificial gene which generates RNA molecule with ribozyme sequences.
Self-catalyzed cleavage to generate the desired gRNA both in vitro and in vivo.
RGR can be transcribed from any type of promoter and thus allow tissue- specific genome editing and efficient
detection of mutations.
Polycistronic tRNA-gRNA (PTG/Cas9)
Array of tandem tRNA-gRNA units, with each gRNA containing a target- specific spacer for simultaneously
targeting multiple sites.
The primary transcript of PTG is cleaved after precise processing via the endogenous tRNA-processing system
by RNase P and RNase Z, which releases numerous mature gRNAs in vivo from a synthetic polycistronic gene.
The excised mature gRNAs direct Cas9 to multiple targets which significantly increase CRISPR/Cas9 multiplex
editing efficiency in plants.
GUIDE RNA VARIANTS
17. • Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining
(NHEJ) or homology-directed repair (HDR) pathways.
• Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable
length at the site of the DSB.
• HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded
or double-stranded DNA donor template.
Sander and Joung 2014 Nature Biotech
18. Cas9 generates DSBs through the combined activity of two nuclease domains, RuvC and HNH. The exact amino acid
residues within each nuclease domain that are critical for endonuclease activity are known D10A for HNH and H840A for
RuvC in S. pyogenes Cas9.
19. • The high rate of alterations created by Cas9, makes easy identification of the desired mutations without
drug-resistance marker selection.
Enhancing Specificity with Cas9 Nickase
• Modified versions of the Cas9 enzyme containing only one
active catalytic domain (called “Cas9 nickase”) bind DNA
based on gRNA specificity, but nickases are only capable of
cutting one of the DNA strands, resulting in a “nick”, or
single strand break, instead of a DSB.
• Two nickases targeting opposite strands are required to
generate a DSB within the target DNA (often referred to as a
“double nick” or “dual nickase” CRISPR system). This
requirement dramatically increases target specificity, since it
is unlikely that two off-target nicks will be generated within
close enough proximity to cause a DSB.
http://www.addgene.org/crispr/guide/
20. Design vectors to express CRISPR/Cas9 in plants
To express gRNA-Cas9 in plants, a plasmid vector (RGE vector) should contain following components :
•RNA Polymerase III (Pol III) promoter and Pol III terminator (Pol III Ter) to control the transcription of gRNA
•gRNA with spacer sequence paired to targeting PAM-site
•RNA polymerase II (pol II) promoter and Pol II terminator (Pol II Ter) to control the transcription of Cas9 gene
•Cas9 with nuclear localization signal (NLS)
Examples of promoters used to construct RGE vector:
•Pol II promoter:
CaMV 35S promoter, maize UBIQUITIN promoter, rice ACTIN promoter, etc.
•Pol III promoter:
snoRNA U3 promoter, snoRNA U6 promoter, etc.
http://www.genome.arizona.edu/crispr/instruction.html
21. (A) Floral dip transformation of Arabidopsis with transgenic T-
DNA carrying Agrobacteria.
(B) Transient inoculation of plant leaf tissue or calli with
Agrobacteria harboring Cas9 and gRNA T-DNA.
(C)Viral vector delivery causes a transiently transformed plant (at
left) to develop systemic infection upon viral capsid replication
after initial transformation of vector DNA.
(D) Transient particle bombardment of plant leaf tissue using a
gene gun with Cas9 and gRNA.
(E) gRNAs only to stable Cas9-expressing transgenic plants.
(F) Ribonucleoprotein (RNP) complex delivery directly to
protoplasts using PEG transformation.
(G) RNA delivery directly to protoplasts (shown here) using PEG
transformation or calli using “gene gun”as in (D).
Lowder et al. 2016 Frontiers in Plant Science
Delivery of CRISPR Reagents to Plant Cells and Tissues
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.
Ishano et al. Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product.
Barrangou et al. Streptococcus thermophilus CRISPR1 locus overview, newly acquired spacers in phage-resistant mutants, and corresponding phage sensitivity.
Zhang lab-The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells.
Doudna lab- reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
The crRNA and tracrRNA are connected by a linker
CRISPR interference. The CRISPR/Cas systems may target either DNA or RNA to interfere with viruses, plasmids, prophages, or other chromosomally encoded sequences.
Model for target search, recognition and cleavage by Cas9:RNA. The search initiates through random 3D collisions. Cas9:RNA rapidly dissociates from non-PAM DNA, but binds PAMs for longer times and samples adjacent DNA for guide RNA complementarity, giving rise to a heterogeneous population of intermediates. At correct targets, Cas9:RNA initiates formation of an RNA:DNA heteroduplex, and R-loop expansion propagates via sequential unwinding. The DNA is cleaved and Cas9:RNA remains bound to the cleaved products.
In comparison to the RNAi concept of transcript degradation and/or translation blocking, the CRISPRi system blocks the transcription initiation and elongation when dCas9/sgRNA is fused with a repressor. Therefore, dCas9/sgRNA system offers a general platform for RNA-guided DNA targeting for stable and efficient modulation of transcription. The dCas9 has been fused to effector domains with distinct regulatory functions for functional mapping of promoters and other genomic regulatory modules.
Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and need to be considered when designing a gRNA for your experiment.
